Immunosuppressive mesenchymal cells and methods for forming same
a technology of mesenchymal cells and mesenchymal cells, applied in the field of immunosuppressive mesenchymal cells and methods for forming same, can solve the problem of not being effective in the hit-in-run paradigm
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[0123]Here we propose a specific in vitro priming protocol of MSCs that can enhance their immunosuppressive qualities. We first compared combinations of three different stimuli: hypoxia (1% O2), IFN-γ (100 ng / mL), and IL-10 (10 ng / mL). Based on gene expression studies for >12 immunosuppressive / protective factors, the conditioning regimen of the combination of IFN-γ and hypoxia for two days led to the target expression of genes known to induce immunosuppression. Those with the greatest fold increase post-stimulation included PDL1, IDO, HLA-G, and COX2, the first two being responsive to the IFN-γ component, while the latter two were mainly upregulated by hypoxia. Importantly, the expression of these genes remained increased over baseline for over one-week post-stimulation, a phenomenon that could be re-capitulated upon re-stimulation.
[0124]Here, we explored combined IFN-γ and hypoxia priming of human MSCs. After 48 h of stimulation, several immunosuppressive factors were upregulated, ...
example 2
[0127]Genes induced by dual priming stay upregulated for up to one week and can be re-induced. To better understand how gene expression changes would persist in the setting of therapeutic application, dual primed MSCs were returned to control conditions, and qRT-PCR for HLA-G, IDO, PD-L1, and COX-2 was performed after 4 days and 7 days. Notably and unexpectedly, for all three MSC donors, HLA-G, IDO, and PD-L1 remained significantly upregulated after being returned to control conditions for 7 days, although a noticeable drop from their peak expression could be seen by day 4 (FIG. 4B). Consistent with the kinetic studies that showed a decline in COX-2 expression by 48 hours, COX-2 expression continued to mildly decline for MSCs that had previously been kept in either control or priming conditions. Since primed MSCs may be re-exposed to inflammatory and hypoxic cues in the patient, the priming regimen was repeated after being returned to control conditions for seven days, and the induc...
example 3
[0128]Dual priming induces immunomodulatory factors at the protein level. In FIG. 6, data are shown for various 48-hour priming regimens. Histograms are from a representative experiment. For clarity, only control MSCs vs. dual primed MSCs are shown on the left, whereas all conditions are shown on the right. The table at the bottom of FIG. 6 shows the mean fluorescence intensity (MFI) of the primed MSCs normalized to the MFI of Control MSCs for n=3 experiments. Significance is shown as (*) for IFN-γ vs. hypoxia stimulation and (t) for IFN-γ vs. dual stimulation. Flow cytometry for HLA-G, IDO, PD-L1, and COX-2 confirmed that they were upregulated at the protein level after dual priming for 48 hours (FIG. 6, top). Considering both single factor and dual factor priming regimens, there were some different patterns at the protein level as compared with the initial PCR findings (FIG. 6, bottom). At the protein level, IDO had slightly more induction by dual priming (although nonsignificant)...
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